The present invention relates to flow cytometry, and more particularly, to a system and method for using multiple lasers in flow cytometry.
In a typical flow cytometer 10, as shown in FIG. 1, a sample solution of particles 12 is combined with a sheath fluid 14. The particles may be fluorescently-labeled, and may be cells or microspheres made of polystyrene or other material. The sheath fluid 14 flows in such a way as to hydrodynamically focus the particle containing sample solution 12 for analysis. The particle containing sample solution 12 and the sheath fluid 14 flow along a flow path 16. An excitation light source 18, typically a laser, is focused upon the particles 12 as they flow along the flow path 16 to induce fluorescence from any reporter dyes present in or on the particles. Any fluorescence from the particles is captured via collection optics 20 positioned orthogonal to the path of the laser beam, and detected using a photomultiplier tube 22.
A forward angle light scatter (FALS) detector 24, typically a photodiode or other light detector, is placed just off the laser axis and captures light scattered by the particle. It is the signal from the FALS detector that indicates the presence of the particles and is usually the trigger for data collection. When the amplitude of the FALS detector signal is greater than a predetermined threshold value, indicating the presence of a particle, data collection electronics are triggered and signals generated by the photomultiplier tubes are acquired as either integral and/or peak values.
For a single laser system, alignment of the laser beam to the flow path, as well as alignment for the collection of the scattered light, is straightforward. Typically, alignment involves adjusting the position of the laser beam to maximize the FALS response, then adjusting the collection optics to maximize the fluorescent signal. This process is illustrated in U.S. Pat. No. 4,038,556, the entire contents of which are hereby incorporated herein by reference.
To facilitate multiplexing, a particle may contain one or more encoding dyes that need to be excited by one or more excitation light sources. The use of multiple excitation light sources generally adds an increased level of complexity, because all excitation light sources need to be aligned with respect to the collection optics.
One solution is to align the excitation light sources so that they focus to the same point in the flow chamber. The excitation light sources may be collinear or not, but should coincide in the detection zone. The mutual alignment of the excitation light sources should be performed by observing the forward scatter signal from each of the excitation light sources as particles pass through the flow chamber using an oscilloscope. The positions of the excitation light sources are adjusted until the forward scatter from the excitation light sources coincides in time. This results in the excitation light sources striking the particle at the same location in the flow chamber. This adjustment is often very cumbersome and time consuming, and any relative misalignment of the excitation light sources may cause signal reduction for one or more fluorescent channels.
It is often desirable to separate the excitation light sources so that each particle passes sequentially through each excitation light source. Separation of the excitation light sources results in a spatial separation in the signals from the particles, which facilitates the capture of the specific responses of the particles to separate excitation sources. However, separation of the excitation light sources adds increased complexity, because the signals are temporally separate. An example of a system employing separated lasers is disclosed in U.S. Pat. No. 4,243,318, the entire contents of which are hereby incorporated herein by reference.
Alternative solutions to temporal separation, such as the use of gated amps or delay lines, require preexisting knowledge of the relative separation of the excitation light sources and cannot correct for excitation light source or core velocity drift during the course of an experiment. Examples of alternative solutions are shown in U.S. Pat. Nos. 5,528,045, 5,682,038, 5,880,474, and Beckman Coulter EPIC 750 and Beckman Coulter ELITE Manuals, the contents of which are hereby incorporated herein by reference.
There is therefore a need for an improved method of aligning two or more excitation light sources with particles in a flow chamber.